Dental caries is one of the most prevalent diseases in the U.S. with 85% of adults and the majority of children having been treated for at least one incidence. While this disease impacts all individuals, it is especially an issue for those of low economic status who have limited access to professional oral care. Dental caries is attributed to the presence of the acidogenic bacterium Streptococcus mutans within biofilms, or plaque, on oral surfaces. Biofilms are surface-attached communities of bacteria that are surrounded by a protective matrix. Bacteria in biofilms are upwards of 1000 times more resistant to currently used antimicrobials than free-floating bacteria. As such, antimicrobials used in oral rinses do not effectively eliminate biofilms; therefore, plaque continues to accumulate, resulting in dental caries. Based on the prevalence and economic disparity of the disease, an affordable, over-the-counter (OTC), widely-distributed solution to plaque removal is needed. In Phase I, Agile Sciences identified five lead anti-biofilm molecules that are effective at dispersing oral biofilms that cause dental caries. These Agilyte molecules are derived from a natural product that controls biofouling of a marine organism. The lead compounds are able to selectively inhibit and disperse biofilms of S. mutans, while not affecting commensal Streptococcal species, S. gordonii and S. sanguinis. Compound efficacy was demonstrated under growth conditions that mimic saliva flow within the oral environment. The lead compounds are non-cidal to bacteria and not cytotoxic to eukaryotic cells at active concentrations. The compounds are also able to disperse multi-species biofilms formed by human saliva on a tooth-like surface. Treatments of saliva-derived biofilms with current OTC oral rinses revealed that the AgilyteTM compounds are able to increase the efficacy of the active ingredients by >2-fold, and this enhancement of oral rinse activity is seven times greater under flow conditions that are more representative of the oral environment. In this Phase II project, we will use in vitro and in vivo analyses to further develop these lead compounds as anti-biofilm additives for OTC oral rinses. In Aim 1, toxicity profiles of the lead compounds will be determined. In Aim 2, an in vivo model of oral colonization will be used to determine if the biofilm reduction capabilities seen in vitro translat to the ability to decrease plaque load in vivo within formulation. A single lead compound identified in Aim 2 will be evaluated in Aim 3 for stability within formulation and will undergo synthetic optimization for large- scale production. The major milestone to be achieved in this Phase II project will be identification of a lead molecule that is non-toxic, active in vivo, and cn be synthesized on a large scale for commercial production. In Phase III, the formulated Agilyte oral rinse will undergo additional preclinical and clinical testing that will facilitate commercialization of an OTC oral rinse product that is highly effective at removing pathogenic biofilms from teeth. This product has the potential to improve personal oral healthcare, thereby decreasing the incidence of dental caries, particularly for those of low economic status that are most affected by the disease. PUBLIC HEALTH RELEVANCE: The incidence of dental caries is staggering with occurrence of the disease being second only to the common cold. Dental caries are caused by pathogenic bacteria within plaque, a mixed community of bacteria known as a biofilm that is difficult to remove from tooth and oral tissue surfaces. This project involves incorporating potent anti-biofilm molecules into oral rinses to effectively remove and kill bacteria within plaque so that th incidence of dental caries will be reduced.